Thiamine diphosphate-dependent enzymes are central to a wide range of technological areas and fields of science. The activity of these enzymes is closely related to the presence of the thiamine diphosphate cofactor (ThDP), which carries out catalysis in its activated form. In this state, the ThDP species has an unusual carbanion in the C2 position of the hetero thiazolium ring. For the ThDP-dependent transketolase (TK) enzyme in particular, this step plays a crucial role in the control of physiological routes, with implications on health/ill conditions for living organisms and the enantioselective synthesis of chemicals at the industrial level. For these reasons, a detailed knowledge on the activation mechanism of ThDP is fundamental. In this work, we present a theoretical study on the water-assisted activation of the ThDP cofactor in Homo sapiens transketolase (hTK). We explore the cofactor activation in the hTK with classical molecular dynamics and hybrid quantum mechanics/molecular mechanics calculations, using three layers, two of them described by high-level ab initio methods at the DLPNO-CCSD(T)/CBS plus B3LYP-D3/6–311 + G(2d,2p):AMBER. The activation of ThDP occurs in one asynchronous, water-mediated proton transfer from C2 to the N4’ atoms and exhibits an activation energy of 5.1 kcal·mol^–1. The Gibbs activation energy for the ThDP activation was −10.3 kcal·mol^–1. Finally, by estimating the electrostatic effect per residue, we rationally identify those that can be mutated to design a much more competent TK enzyme.